Conventional optical spectrometers, such as infrared spectrometers, are dispersive instruments, employing a prism or a grating to separate the radiation emitted by the source into its component wavelengths, with each resulting spectral element detected individually. As a result, these spectrometers are inefficient in their use of the energy available from the source since, at any given time, only a small fraction of the energy reaches the detector. This inefficiency is particularly disadvantageous in applications which involve low energy throughput, such as attenuated total reflectance (ATR) experiments. Conventional spectrophotometers utilize filters to block all wavelengths except the wavelength band of choice and, accordingly, also utilize only a fraction of the source energy.
A Fourier transform infrared (FTIR) spectrometer operates on a totally different optical principle of interferometry. An interferometrically coded signal contains information for a range of wavelengths; computing the Fourier transform of the signal yields a spectrum. The first interferometer was built by Michelson in 1891, and his design is incorporated in most commercial FTIR spectrometers currently in use. A corner cube interferometer is also utilized in some commercial FTIR spectrometers.
The most important factor, in practice, contributing to the sensitivity of Fourier transform optical spectrometry (FTOS), which includes FTIR spectrometry, is Fellgett's advantage, or the multiplex advantage, which gives rise to improvements in signal-to-noise ratio (S/N) and reduction in measurement time for FT spectral acquisition, compared to the time required to obtain spectra with a grating instrument. Fellgett's advantage originates in the fact that information for all wavelengths emitted from the source reaches the detector at the same time in FT measurements, whereas in a grating instrument each spectral element (.nu.cm.sup.-1, corresponding to the resolution) is detected separately. This results in a dramatic decrease in measurement time if information from a large spectral region is required. This is an important consideration if multiple components absorbing in different regions of the spectrum are to be detected.
Another advantage associated with FTIR spectrometers is Jacquinot's advantage. The magnitude of Jacquinot's advantage is assessed by comparison of the maximum optical throughput permissible before loss of resolution is incurred for a grating spectrometer and an interferometer. In a grating instrument the throughput is significantly limited by the size of the slits, resulting in low throughput. Since there are no slits in an interferometer, more energy impinges on the detector.
The use of a laser in an interferometer to trigger digitization of the signal gives rise to a third advantage, known as Connes' advantage. This refers to the precision (0.003-0.006 cm.sup.-1) with which frequencies can be determined in FTIR work, because the laser serves as an internal wavelength calibration standard. Since the digitized spectra are stored as a series of data points corresponding to fractions of laser wavelength (which does not vary with time), spectra recorded at widely separated times can be compared with precision. This is a particularly important advantage for data handling techniques such as spectral substraction and coaddition of scans, which can be subject to difficulties when performed on a computerized grating instrument due to drifts in frequency with time.
FTIR spectrometers have been found to be very useful in attenuated total reflectance (ATR) experiments, wherein light from the source is transmitted down a suitable waveguide (the internal reflection element, or IRE), in such a manner that it is totally reflected at the IRE-sample interface, giving rise to an evanescent wave which penetrates into the sample. As a result of the attenuation of the evanescent wave by the sample, the light exiting the IRE and striking the detector is attenuated at wavelengths corresponding to absorptions of the sample, thus yielding the spectrum of the sample. Due to the shallow depth of penetration of the evanescent wave into the sample, a short effective pathlength is obtained. However, the use of ATR techniques with conventional spectrometers is limited by the poor signal-to-noise ratio of the spectra obtained. As a result of the advantages of FT spectrometers, as outlined above, the coupling of ATR techniques with FTIR has made possible the acquisition of high-quality spectra. Several manufacturers have designed small ATR cells offering automatic sampling and self-cleaning capabilities, facilitating the routine quantitative analysis of samples.
RE 33064 (Carter et al.) discloses a method for the determination of an analyte in solution by its reaction with an appropriate reactant coated on the surface of an optical waveguide. The basis of the method is the detection of the modification of a multiply totally reflected light wave travelling through the waveguide core by the formation of a layer of the analyte-reactant product on the waveguide surface. The application of this technique in immunoassay, i.e., the case in which the analyte is an antigen and the reactant is an antibody, or the reverse condition, is included in the teachings of this patent.
It would be highly desirable if to design a system wherein an interferometrically coded signal from an infrared source would be propagated down an optical waveguide, and the resulting attenuation of the evanescent wave would provide a measure of the amounts of an antigen-antibody complex bound on the outer surface of the waveguide.